Regulation of Staphylococcus aureus colonization and disease

项目摘要 金黄色葡萄球菌是一种条件致病菌，可引起多种急性和慢性疾病。 感染。抗生素耐药性水平正在上升，耐甲氧西林金黄色葡萄球菌(MRSA)感染 更具挑战性的治疗，导致患者和医疗系统的负担增加。尽管 这种有效的病原体金黄色葡萄球菌可以无症状地定植在大约20%的健康成年人身上。 人口，主要是在鼻腔，其次是皮肤。显然有必要了解 从殖民者到入侵者的转变，因为大多数金黄色葡萄球菌疾病是由来自 殖民菌株。我们最近的发现表明，ArlRS TCS在金黄色葡萄球菌的转变中起着关键作用 一种侵袭性病原体。ArlRs的主要输出是控制细胞质MGRA的表达 抑制尿素酶和大表面蛋白并诱导免疫逃避因子的调节剂。我们的模型是 金黄色葡萄球菌在MGRA水平较低时表现出共生的特征，并表现出入侵的特征 当MGRA水平较高时，病原体。为了测试这个模型，在目标1中，我们将确定ArlRS对 金黄色葡萄球菌的黏附、定植和感染。为此，我们将比较WT、∆ARLRS和∆MGRA 使用体外黏附模型和体内皮肤定植模型的突变菌株。此外，我们还将 进行实时体内成像，跟踪MGRA调节子的表达，进行皮肤感染和免疫 对这些菌株的逃避评估。在目标2中，我们将对ArlS和ArlS进行生化表征 识别依赖ArlR的启动子。ArlR是OmpR家族的反应调节因子，而ArlR DNA- 结合位点和目标启动子未知。ArlS激酶有一个N-末端的传感器结构域，带有两个 膜跨越通过侧翼的14.1 kDa细胞外缓存结构域。这一目标的目标是 测定ArlS和ArlR的生化性质，将为深入了解ArlS和ArlR的作用机制提供重要的依据 ArlS调控和ArlR启动子靶点。我们将评估ArlS-ArlR磷酸转移的动力学 突变体，以确定酶活性(激酶和磷酸酶活性)，这是调节 ArlRS规则。我们还将使用SELEX来鉴定ArlR结合位点，并用qRT-确认目标启动子 聚合酶链式反应和RNAseq.在目标3中，我们将对ArlS传感机制进行分子分析。我们假设 ArlS细胞外缓存域对导致高尿素酶和SasG的环境提示做出反应 促进金黄色葡萄球菌定植的表达。为了进一步研究这种机制，我们将研究Signal 使用报告菌株控制ArlS功能，我们将在ArlS缓存域中进行站点定向更改 残留物和评估功能。我们还将从小分子中筛选额外的ArlS Cache域配体 文库采用热位移聚合酶链式反应和核磁共振技术。发现改变ArlRS功能和预防的配体 金黄色葡萄球菌转变为侵袭性病原体可能在治疗抗生素耐药感染方面具有潜力。

Project Summary Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance levels are growing and methicillin-resistant S. aureus (MRSA) infections are more challenging to treat, resulting in increased burden on both patients and healthcare systems. Despite being such an effective pathogen, S. aureus can asymptomatically colonize approximately 20% of the healthy adult population, primarily in the nasal cavity and secondarily on the skin. There is a clear need to understand the transition from colonizer to invader, since the majority of S. aureus disease is the result of autoinfection from the colonized strain. Our recent findings indicate that the ArlRS TCS plays a critical role in the S. aureus transition to an invasive pathogen. The primary output of ArlRS is controlling the expression of MgrA, a cytoplasmic regulator that represses urease and large surface proteins and induces immune evasion factors. Our model is that S. aureus exhibits the traits of a commensal when MgrA levels are low, and exhibits the trait of an invasive pathogen when MgrA levels are high. To test this model, in Aim 1 we will determine the contribution of ArlRS to S. aureus adherence, colonization, and infection. Toward this end, we will compare WT, ∆arlRS, and ∆mgrA mutant strains using in vitro models of adherence and an in vivo model of skin colonization. Additionally, we will perform real-time in vivo imaging to track MgrA regulon expression, and carry out skin infection and immune evasion assessments of these strains. In Aim 2, we will perform a biochemical characterization of ArlS and identify of ArlR-dependent promoters. ArlR is a response regulator of the OmpR family, and the ArlR DNA- binding site and target promoters are unknown. The ArlS kinase has an N-terminal sensor domain with two membrane-spanning passes that flank a 14.1 kDa extracellular Cache domain. The goal of this aim is to determine the biochemical properties of ArlS and ArlR, which will provide critical insight into the mechanism of ArlS regulation and ArlR promoter targets. We will assess the kinetics of ArlS-ArlR phosphotransfer using mutants to determine the enzyme activity (kinase and phosphatase activity) that is important for regulating the ArlRS regulon. We will also use SELEX to identify the ArlR binding site and confirm target promoters with qRT- PCR and RNAseq. In Aim 3, we will perform molecular analysis of the ArlS sensing mechanism. We hypothesize that the ArlS extracellular Cache domain responds to an environmental cue that results in high urease and SasG expression to promote S. aureus colonization. To further investigate this mechanism, we will examine signal control over ArlS function using reporter strains, and we will make site-directed changes in ArlS Cache domain residues and assess function. We will also screen for additional ArlS Cache domain ligands from small-molecule libraries by thermal shift PCR assays and NMR. Discovering ligands that alter ArlRS function and prevent transition of S. aureus to an invasive pathogen could have potential in treating antibiotic-resistant infections.